The high rate of mutation and genetic rearrangement, particularly for the surface antigens hemagglutin and neuraminidase, results in annuals shifts in the predominant flu viruses and periodic flu pandemics that can threaten entire populations. Diagnostic methods are therefore needed to identify and to protect the world's populations against these emerging new strains. In recent years viral diagnostics has been rapidly shifting to nucleic acid testing (NAT) because of the power of such methods for detecting underlying genomic changes that are the cause of antigenetic changes and that enable precise discrimination between species and strains. NAT diagnostics has primarily focused on PCR-based methods, particularly real-time PCR, because these methods offer sample amplification and speed. However, PCR-based methods are limited to a few targets and real-time PCR suffers from high rates of false positives and false negatives. The investigator has developed novel universal probes that can easily be applied to any target at low cost and that provide the capacity to detect multiple targets in one tube with a combined signal. This technology also enables multi-target, multi-color detection which provides distinctive signaling patterns. These new features can overcome both false negatives and false positives, thereby increasing assay reliability. This application plans to extend and optimize development of prototype universal probes for H5, H3 and M1 of flu, and to create additional probes for H1 and flu B. Tests will also be conducted to compare the performance of the new universal probes against standard Taqman probes directed to the same targets. The expected outcome of these innovative methods should produce rapid, sensitive, and low cost universal probe systems for real-time influenza detection and discrimination. PUBLIC HEALTH RELEVANCE: The development program combines a proven nucleic acid amplification platform with new probe technologies invented by the investigator that enable simultaneous, low cost detection of multiple target sites in a pathogenic species. This technology can thus supplant current Taqman probes which are costly and detect only one sequence target. Multi-target detection prevents false negative tests and improves reliability. False positives can also be avoided by a variation of the technology that enables multi-color detection. The probes will detect and discriminate the major strains of influenza A by detecting 3 target sites in H5, H3, H1, and MI. These diagnostic probes are intended for commercial distribution in hospital and clinical labs for testing and monitoring infectious disease and for managing treatment.